Experimental Research On Cloning, Constructing Of Prokaryotic Expression Vector And Protein Expression About Kringle5

Background & Objective:The one of the key links of the tumor growth and metastasis is angiogenesis,thus blocking or inhibiting angiogenesis is an effective anti-tumor therapeutic strategy.In recent years,the formation of the vascular mechanism has been extensively and deeply researched,and it has been proved by a variety of angiogenesis-related factor of adjustment.People have found a variety of angiogenic factors and angiogenesis inhibitor factors.Angiogenesis as a target in the treatment of tumors has become a research point in cancer therapy,there are a variety of angiogenesis inhibitors have entered clinical trials.Human Plasminogen Kringle5(referred to as K5) is a newly discovered angiogenesis inhibitor.It can act on the proliferation of vascular endothelial cells, inhibit cell proliferation,migration,and induction of apoptosis,and it is the most powerful endogenous inhibitor of angiogenesis.As the effect of k5 are a inhibiting endothelial cell proliferation and anti-migration are strong,the value of application of K5 is prominent.At present,gaining K5 mainly through genetic engineering of recombinant technology.Its purification is complex,yield is low,and the relatively molecular weight of K5 is large.Therefore,we clone a simplified K5 directly and carry out fusion expression with GST by studying the function regional and spatial structure of K5.The purpose of this experiment is to find a quick and efficient way to gain the K5 protein,laying foundation for the development of anticancer drugs.Method:To extract total RNA from the human bladder cancer tissue,designing a pair of rational primers,we gain the K5 by RT-PCR.Cloning the gene to prokaryotic expression plasmid pGEX-5X-1,then this recombinant vector was transformed into E. coli BL-21.To identify the positive clones by PCR and restriction enzyme digestion, to inducing positive clones with IPTG,we has successfully gained the fusion proteins, then to observe the expression products with SDS-PAGE.Results:Successfully,the prokaryotic expression vector(K5/pGEX-5X-1) was constructed,and the K5 fusion protein was harvested by inducing the expression of BL-21.Conclusion:K5 exogenous protein express successfully in prokaryotic cells.It provides a practical method for gaining the K5 protein.This laid the foundation for follow-up experiment to explore the activity of protein research and the preparation and purification,K5 on the inhibition of vascular endothelial cells,and also it could be an novel method on the study of tumor growth in animal models,Inhibition of tumor invasion and migration research,as well as the development of anti-tumor drugs.